Hyperhomocysteinemia is an independent risk for cardiovascular disease. Most of the reported biological effects of homocysteine (Hcy) in vascular cells have been attributed to oxidative mechanisms, which were observed at Hcy concentrations higher than 1mM, and can be mimicked by cysteine, another non-pathogenic biothiol. Thus, a biochemical mechanism unique to Hcy remains to be identified. Our previous work has demonstrated that Hcy at 10-50 muM, but not cysteine, arrests endothelial cell (EC) p21/ras, suppress cyclin A transcription in cell type specific manner. The basic hypothesis of this proposed project is that Hcy, at growth through a hypomethylation related mechanism, which blocks cell cycle progression and endothelium regeneration. This project will study this hypothesis utilizing three linked specific aims. First, in Aim 1, experiments are designed to elucidate the role of Ras demethylation-independent Ras over-expression on EC growth, and on cyclin A expression and promoter activity. Cell growth will be determine by thymidine uptake, flow cytometry and cell counting. Cyclin A expression and promoter activity will be determined by Northern, Western blot analysis and reporter gene transfection. Alternatively, DNA microarray will be used to identify other potential targets in Hcy signaling. Second, in Aim 2, studies are proposed to determine the biochemical mechanisms by which Hcy suppress cyclin A transcription. Experiments will be performed to study the RB phosphorylation and E2F expression, and the role of E2F and other transcription factors on cyclin A transcription by Western blot, reporter gene transfection, gel mobility shift, adenovirus-transduced E2F expression. The role of cyclin A in maintaining RB phosphorylation will be assessed by RNA interference. The promoter methylation pattern of cyclin A genes will be examined by bisufite genomic sequence and methylation sensitive restriction enzyme digestion. Finally, in Aim 3, a mouse endothelial denudation and regeneration model will be used in cystathionine beta-synthase (CBS) knockout and diet-induced hyperhomocysteinemic mice to assess the effect of Hcy in endothelial regeneration. Mouse blood will e examined for the concentrations of Hcy and SAH/SAM. Immunohistochemistry staining for leukocytes, macrophages, EC and smooth muscle cells will be performed on vessel sections to analyze the cellular composition of the lesion. In situ hybridization of immunohistochemistry staining for cyclin A will be performed to assess its involvement. The broad, long-term objective of this proposal i to elucidate Hcy signaling in EC growth inhibition, and to evaluate its importance on the role of atherogenesis in hyperhomocysteinemia. If we identify the key events in Hcy-induced arteriosclerosis, genetic or biochemical approaches to block these steps could lead to therapeutic advantage.